Porous membrane having immobilized enzyme, porous membrane composite including the same, and preparation method thereof

ABSTRACT

Disclosed herein is a porous membrane having an immobilized enzyme wherein the enzyme is immobilized within pores which are three-dimensionally connected to each other. The porous membrane having the immobilized enzyme is three-dimensionally crosslinked in a molecular level wherein nanopores of 5 to 100 nm are interconnected, so that the immobilized enzyme may be in contact with a reactant in all directions, and the reaction solution may be easily diffused, thereby proceeding with the catalytic reaction fast and conveniently without deterioration of material transport.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to Provisional Application Ser. No.61/887,482 filed on 7 Oct. 2013, and Korean Patent Application No.10-2014-0006681 filed on 20 Jan. 2014, and all the benefits accruingthere from under 35 U.S.C. § 119, the contents of which is incorporatedby reference in its entirety.

TECHNICAL FIELD

The present invention relates to a porous membrane having an immobilizedenzyme, and more particularly, to a porous membrane which is an organicporous monolithic organic reticular membrane, three-dimensionallycrosslinked in a molecule level, including an immobilized enzymetherein, and a preparation method of the membrane.

BACKGROUND

An enzyme generally has high three-dimensional and chemical selectivity,thereby being useful in various reactions, and is used as a catalystaccelerating a reaction rate even under a mild reaction condition.However, since enzymes are generally costly, they have an economicproblem in use in a large amount in an industrial scale. In addition,since most enzymes are insoluble in an organic solvent, they are limitedin use in an organic chemical reaction. Therefore, in order to improveactivity and stability of the enzyme and reuse the enzyme, research toimmobilize the enzyme has much proceeded.

There are largely three ways to immobilize an enzyme on a polymermembrane. The first one is to adsorb an enzyme on a surface of a polymermembrane, the second one is to modify an enzyme to adhere it on apolymer membrane by a covalent bond, and the last one is an entrappingmethod to physically trap an enzyme in pores of a polymer membrane.Since it is the easiest and simplest way to adsorb an enzyme on asurface of a membrane by a non-covalent bond, much research thereon hasbeen done. However, the enzyme may be easily separated, and thestability of the enzyme is relatively low. Modifying an enzyme andadhering it on a membrane by a covalent bond is the way to most stablyimmobilize an enzyme. However, since a process of modifying an enzyme isaccompanied, the activity of an enzyme is lowered, and a process ofimmobilizing an enzyme is complicated.

The following patent document 1 relates to a method of immobilizing anenzyme using a double template, wherein the enzyme is immobilized on thedouble template to show a high catalytic activity. However, thepreparation procedure of the catalyst is complicated, and theimmobilized enzyme blocks pores, thereby making mass transfer difficult.

RELATED ART DOCUMENT Patent Document

(Patent document 1) Korean Patent Laid-Open Publication No. 2011-0029748

SUMMARY

An object of the present invention is to provide a porous membranehaving an immobilized enzyme, which has high immobilization rate andimmobilization retention rate of the enzyme, long-term stability of theenzyme activity, and has excellent mass transfer capability.

Another object of the present invention is to provide a preparationmethod of the porous membrane having an immobilized enzyme, wherein theprocess of immobilizing the enzyme is simple.

According to an exemplary embodiment of the present invention, there isprovided a porous membrane having an immobilized enzyme, wherein theporous membrane is three-dimensionally interconnected by pores, thepores have a size of 5 to 100 nm, and the porous membrane forms athree-dimensional network by polymerization of a monomer having 2 to 4amino groups and a monomer having 2 to 4 isocyanate groups.

According to an embodiment of the present invention, the porous membranemay be a flat membrane or a hollow fiber membrane.

According to an embodiment of the present invention, the enzyme may beone or more selected from the group consisting of lipase, amylase,protease, trypsin, papain, brinase, peroxidase, horseradish peroxidase(HRP), carbonic anhydrase, aquaporin, chymotrypsin, subtilisin, soybeanperoxidase, chloroperoxidase, manganese peroxidase, tyrosinase, laccase,cellulase, xylanase, lactase, sucrase, organophosphohydrolase,chlorinesterase, glucose oxidase, alcohol dehydrogenase, glucosedehydrogenase, hydrogenase, and glucose isomerase.

According to another exemplary embodiment of the present invention,there is provided a preparation method of the porous membrane having animmobilized enzyme, wherein the porous membrane is penetrated with asolution containing the enzyme.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow chart representing a process of preparing the porousmembrane having the immobilized enzyme of the present invention.

FIG. 2(a) is a graph representing nitrogen adsorption/desorption curvesof the porous membrane having immobilized lipase according to anexemplary embodiment of the present invention, and FIG. 2(b) is a graphrepresenting a pore size distribution of the porous membrane havingimmobilized lipase according to an exemplary embodiment of the presentinvention.

FIG. 3 is a schematic diagram of the preparation method of the membranehaving the immobilized enzyme of the present invention.

FIG. 4 is a FT-IR spectrum of the porous membrane prepared according toan exemplary embodiment of the present invention.

FIG. 5 is a confocal microscopic image of the porous membrane preparedaccording to an exemplary embodiment of the present invention.

FIG. 6 is a graph representing an amount of an enzyme immobilized on theporous membrane over time.

FIGS. 7(a) to 7(c) is a graph representing the result of measuring eachsolution by a gas chromatography in Example 2.

FIG. 8 is a SEM (scanning electron microscopic) image representing theporous membrane prepared according to an exemplary embodiment of thepresent invention before and after removal of polyethylene glycol (PG)from the membrane.

FIG. 9 is a graph representing an oleic acid conversion rate overpenetration time of the porous membrane prepared according to anexemplary embodiment of the present invention.

FIG. 10 is a graph representing an oleic acid conversion rate accordingto the number of reuse of the porous membrane prepared according to anexemplary embodiment of the present invention.

DETAILED DESCRIPTION OF EMBODIMENTS

Hereinafter, the present invention will be described in detail. Herein,it should be understood that the terms such as “first” and “second” areused not for limiting, but for distinguishing the constituents of theinvention.

The porous membrane having an immobilized enzyme according to thepresent invention includes a porous membrane forming a three-dimensionalreticular nanopores, and an enzyme captured within pores of the porousmembrane.

The porous membrane forms a three-dimensionally cross-linked monolith,and includes pores having a size of 5 to 100 nm, wherein the pores areinterconnected, so that the enzyme immobilized therein may be in contactwith a reactant in all directions, and the solution may be easilydiffused, and thus, reduction in material transport, caused by theenzyme blocking pores, does not occur.

Further, the size of pores of a porous carrier for immobilizing anenzyme therein is generally known to be 20 to 50 nm (Membrane-BasedSynthesis of Nanomaterials, Charles R. Martin), and since the porousmembrane of the present invention has wide range of nanopores of 5 to100 nm, it may immobilize the enzyme having various sizes.

The enzyme which may be immobilized in the porous membrane of thepresent invention may be specifically a digestive enzyme such as lipase,amylase or protease, a proteolytic enzyme such as trypsin, papain orbrinase, or the like, and peroxidase, horseradish peroxidase (HRP), orthe like may be used for water treatment. Further, carbonic anhydrasemay be used for capturing carbon dioxide. Additionally, the enzyme maybe one or more selected from the group consisting of aquaporin,chymotrypsin, subtilisin, soybean peroxidase, chloroperoxidase,manganese peroxidase, tyrosinase, laccase, cellulase, xylanase, lactase,sucrase, organophosphohydrolase, chlorinesterase, glucose oxidase,alcohol dehydrogenase, glucose dehydrogenase, hydrogenase, and glucoseisomerase, but not limited thereto.

The porous membrane of the present invention may be obtained by mixingan organic sol consisting of an organic reticular structure obtained bypolymerizing a first monomer having an amino group and a second monomerhaving an isocyanate group, an acyl halide group or an ester group whichis a functional group polymerizable with the amino group, with a polymerto produce a porous membrane via phase separation into the polymer andorganic sol phases and the subsequent gelation of organic sol phase, andremoving the polymer from the porous membrane using a solvent thatdissolves the polymer.

According to an embodiment of the present invention, the first monomerhas 2 to 4 amino groups, and the second monomer has functional groupsselected from the group consisting of 2 to 4 isocyanate groups, acylhalide groups and ester groups.

The first monomer having 2 to 4 amino groups may be an aliphaticcompound having 1 to 100 carbons, substituted with 2 to 4 amino groups,or an aromatic compound having 6 to 100 carbons, substituted with 2 to 4amino groups.

The second monomer having 2 to 4 isocyanate groups, acyl halide groupsor ester groups may be an aliphatic compound having 1 to 100 carbons,substituted with 2 to 4 isocyanate groups, acyl halide groups or estergroups, or an aromatic compound having 6 to 100 carbons, substitutedwith 2 to 4 isocyanate groups, acyl halide groups or ester groups.

The first and second monomers may be, as an example, compoundsrepresented by the following Chemical Formulae 1 to 9:

wherein R is an amino group, an isocyanate group, an acyl halide group,or an ester group.

Further, according to an embodiment of the present invention, the firstand second monomers may be the compound represented by the followingChemical Formula 10:

wherein R is an amino group, an isocyanate group, an acyl halide group,or an ester group; and n is 0 or 1.

The first monomer and the second monomer are polymerized by reactionbetween the amino group of the first monomer and the isocyanate group,the acyl halide group or the ester group of the second monomer, togenerate a crosslinked network.

That is, the organic reticular structure formed by the polymerizationbetween the first monomer and the second monomer is three-dimensionallypolymerized and crosslinked to have micropores and a large specificsurface area, and has excellent chemical resistance, thermal resistanceand durability by a high crosslinking rate and a strong covalent bond.

Further, the monomer having 2 to 4 amino groups may be, as an example,tetratis(4-aminophenyl)methane (TAPM), p-phenylene diamine (PDA), oroxydianiline (4,4′-oxydianiline) (ODA), but not limited thereto.

Further, the monomer having 2 to 4 isocyanate groups may be, as anexample, p-phenylene diisocyanate (PDI), hexamethylene diisocyanate(HDI), or tetrakis(4-isocyanatophenyl)methane (TIPM), but not limitedthereto.

According to an embodiment of the present invention, the porous membranemay be formed by polymerization of the monomer represented by thefollowing Chemical Formula 11 and the monomer having 2 isocyanategroups:

wherein X is a carbon or silicon atom.

Further, according to another embodiment of the present invention, theporous membrane may be formed by polymerization of the monomer having 2amino groups and the monomer represented by the following ChemicalFormula 12:

wherein X is a carbon or silicon atom.

The porous membrane may have a structure of a flat membrane or a hollowfiber membrane.

Further, the present invention provides a preparation method of a porousmembrane having an immobilized enzyme, including (B) passing a solutioncontaining the enzyme through the porous membrane, wherein the enzyme isimmobilized within the pores of the porous membrane.

When the porous membrane is penetrated with the solution containing theenzyme, the solution passes through the porous membrane, and in thecourse of which the enzyme flowing along with the solution is capturedin the pores having a similar size within the membrane, by gravity oroptionally applied pressure. The thus-captured enzyme is physicallyimmobilized by a noncovalent bond with the porous membrane.

The passing of the solution may be carried out in a manner selected fromdead-end flow, cross flow filtration, or the combination thereof, andthe immobilization rate of the enzyme may be further increased byapplying pressure.

According to an embodiment of the present invention, the immobilizationlevel of the enzyme may be much raised by additionally passing a solventfor enzyme immobilization through the porous membrane having theimmobilized enzyme. The solvent for enzyme immobilization may be water.

However, such additional step does not necessarily have to be carriedout separately, and for example, in case where an enzyme for preparingbiodiesel is immobilized, biodiesel may be prepared without an outflowof the enzyme, by including a raw material of biodiesel in the solventfor enzyme immobilization and passing it. Such preparation method of theporous membrane may include (A) obtaining the porous membrane, before(B).

Said (A) may include the following: (a-1) polymerizing the monomerhaving 2 to 4 amino groups and the monomer having 2 to 4 isocyanategroups, acyl halide groups or ester groups to obtain an organic sol,(a-2) adding a polymer solution to the organic sol to obtain a mixedsolution, (a-3) applying the mixed solution onto a substrate then curingit to obtain a network/polymer composite membrane, and (a-4) passingsolvent through the microporous membrane or immersion of the membraneinto solvent to remove polymers, thereby forming a porous membraneincluding nanopores.

The polymer solution is prepared by dissolving a thermoplastic polymerin a suitable solvent such as DMF, DMAc, NMP, DMSO, THF and ethanol, andthe polymer may be any one selected from the group consisting ofpolyethyleneglycol, polysulfone, polyethersulfone, polyacrylonitrile,polyimide, polyetherimide, polybenzimidazole, polymethylmethacrylate,polystyrene, polyetheretherketone and polyvinylidenefluoride.

The pore size and the microstructure of the porous membrane areadjustable according to the amount of the polymer solution added to theorganic sol in above (a-2). As the amount of the polymer solution isincreased, the phase separation between the organic network sol and thepolymer solution much proceeds, thereby making the pores larger.

The solution containing the organic reticular structure formed bypolymerization of the first monomer having 2 to 4 amino groups and thesecond monomer having 2 to 4 isocyanate groups, acyl halide groups orester groups, may proceed with gelation, as it has higher degrees ofpolymerization and crosslinking. However, since a gelation rate dependson the concentration of the mixed monomers, the solution is controllableto the organic sol state which is an intermediate state before gelation,by properly adjusting the concentration of the monomer solution.

Therefore, in the preparation of the porous membrane (A), the resultingporous membrane may have various structures and sizes, by a gelationreaction and a phase separation phenomenon between the organic sol andthe polymer solution. The structure of the porous membrane iscontrollable in the course of preparing the organic sol by the factorssuch as polymerization time of the monomers, the property of theprepared organic reticular structure (a covalent bonding or physicalbonding property within the organic network), the kind and the molecularweight of the polymer added to the sol, and the compositional ratiobetween the organic network and the polymer. Therefore, by properlyadjusting such factors, the porous membrane having a desired propertydepending on its use may be selectively prepared.

In above (a-3), the solvent may be removed from the mixed solutionbefore the mixed solution is applied on the substrate. On removing thesolvent from the mixed solution, the gelation may slowly proceed,thereby making the size of the pores larger.

Further, in above (a-3), the mixed solution may be applied by properlyselecting one of the solution processes such as spin coating, dipcoating, spray coating, casting and doctor blade coating, consideringthe viscosity of the mixed solution and the like.

Further, the present invention provides a preparation method ofbiodiesel, including carrying out a reaction in a reaction solutioncontaining (a) a biodiesel raw material, (b) the porous membrane, and(c) a solvent.

The biodiesel raw material may be a mixture of soybean oil and ethanol,the enzyme immobilized within the porous membrane may be lipase, and thesolvent may be an organic solvent.

Hereinafter, the present invention is illustrated by the followingpreferred Examples, and the like, in more detail. However, thoseExamples and the like are intended to describe the present invention inmore detail, and it will be evident to a person skilled in the art thatthe scope of the present invention is in no way limited thereby.

EXAMPLES Example 1: Preparation of Porous Membrane

(1) Preparation of (TAPM+HDI/PEG) Nanocomposite Membrane

Tetrakis(4-aminophenyl) methane (TAPM, MW:382.50) was dissolved in DMF(N,N-dimethylformaide) to prepare an organic solution having aconcentration of 4 wt/vol %, and 1,6-diisocyanatohexane (HDI;hexamethylene diisocyanate, MW:168.19) was dissolved in DMF to preparean organic solution having a concentration of 4 wt/vol %. Then, thetetra(4-aminophenyl) methane solution was slowly injected to the1,6-diisocyanatohexane solution and mixed. The above mixed solution wasreacted at room temperature under nitrogen atmosphere for 72 hours toobtain a sol-phase mixed solution.

After adding polyethylene glycol (PEG) to the mixed solution in aconcentration of 60 wt % and sufficiently stirring it, the mixture wasapplied on a glass plate, and dried and cured at 50° C. for 1 hour, at80° C. for 2 hours, and at 100° C. for 3 hours, thereby finallysynthesizing the nanocomposite membrane of the organic molecular network(TAPM+HDI) and PEG.

(2) Preparation of TAPM+HDI Porous Membrane—Removal of PEG

After the synthesized membrane was cooled down at room temperature, itwas precipitated in water to be separated from the substrate, andstirred in water for about one week to remove polyethylene glycol (PEG)which is water-soluble polymer. Thus, finally the porous membrane havingnanopores according to the present invention was prepared. The SEMimages of the membrane before and after removing polyethylene glycol areshown in FIG. 8. As shown in FIG. 8, it was confirmed that the porousmembrane was formed by leaving voids in the place where PEG had beenlocated, after removal of PEG.

(3) Immobilization of Enzyme

The porous membrane having an immobilized enzyme of the presentinvention was prepared using lipase as the enzyme.

After preparing a solution formed by dispersing lipase in water (90mg/L), undissolved lipase was removed using a cellulose acetate membranefilter of 0.22 μm, then the porous membrane prepared in above (2) waspenetrated with the thus prepared solution in a manner of dead-end flow,thereby preparing the porous membrane having immobilized lipase.

Confirmation Example 1: Measurement of Pore Size of Membrane HavingImmobilized Lipase

The presence, size and distribution of the pores formed by removing PEGfrom the composite membrane consisting of TAPM/HDI and PEG wereconfirmed through a specific surface area analyzer. FIG. 2(a) is a graphrepresenting nitrogen adsorption/desorption curves, and FIG. 2(b) is agraph representing a pore size distribution. As shown in FIG. 2(b), itwas confirmed that the pores formed by removing PEG which is a polymermatrix had various sizes of 5 to 100 nm, and an average size of 20 nm.

Confirmation Example 2: FT-IR Measurement

FIG. 4 is a FT-IR spectroscopy (Fourier Transform Infrared Spectroscopy)spectrum of lipase, the porous membrane before immobilizing the enzyme(lipase), and the porous membrane having the immobilized enzyme (lipase)prepared according to an exemplary embodiment of the present invention.The membrane prepared according to the present invention (c) has abroader peak at 3350 cm⁻¹ than the membrane before immobilizing lipase(b), which is resulted from an amino group (—NH) of amino acid in lipasewhich is the immobilized enzyme. Further, the peak at 2410 cm⁻¹ wascaused by a carboxyl group (—COOH) of lipase, and the increased peak at670 cm⁻¹ was caused by a CH group in an alkyl chain of lipase. Thus, itis confirmed that the porous membrane of the present invention hasimmobilized lipase which is the enzyme.

Confirmation Example 3: Confocal Microscopic Image Measurement

In order to confirm whether the enzyme was dispersed and immobilizedinside the porous membrane prepared in one exemplary embodiment of thepresent invention, the membrane was applied with green fluorescentprotein, and examined by a confocal microscope. Each image in FIG. 5 isa photograph taken by focusing at a point 40, 35, 25, 15 and 5 μm awayfrom the membrane surface of the present invention, respectively. As thefocusing point changes, the fluorescent protein which at first had beenobserved became invisible, and the fluorescent protein which at firsthad not been observed became visible. This means that the fluorescentproteins were distributed evenly throughout the porous membrane,respectively.

Experimental Example 1: Measurement of Amount of Immobilized Enzyme

The amount of the enzyme immobilized in the membrane prepared accordingto an exemplary embodiment of the present invention was measured using aBCA kit. FIG. 6 is a graph representing an amount of an enzymeimmobilized in the porous membrane over time. As shown in FIG. 6, aspenetration time increased, the amount of the immobilized enzyme alsoincreased. As a result of measuring the absorbance with the solutiontaken after penetration of the enzyme solution for 10 hours, theconcentration of the enzyme solution was reduced by about 40% ascompared with the initial solution, and in case of the solution takenfor 35 hours, the concentration was reduced by about 60% as comparedwith the initial solution. This means that more than about 40% of theenzyme of the solution was immobilized in the porous membrane afterpenetration time of 10 hours, and about 60% of the enzyme of thesolution was immobilized in the porous membrane after penetration timeof 35 hours.

Experimental Example 2: Measurement of Enzyme Activity and Stability

In order to measure the catalytic activity of the enzyme immobilized inthe porous membrane prepared according to an exemplary embodiment of thepresent invention, oleic acid and butanol were used as reactants. Theoleic acid and butanol are converted to butyloleate by esterification.The conversion rate of the oleic acid was calculated by titrating themixture of oleic acid and butanol which is the starting material, andthe solution penetrating the porous membrane of the present inventionwith a potassium hydroxide solution, and the activity of the enzyme wasmeasured using the conversion rate.

Further, it was confirmed that the oleic acid conversion rate was wellmaintained on continuous operation of the membrane for a long time bypassing the mixture of oleic acid and butanol through the porousmembrane having the immobilized enzyme of the present invention. FIG. 9is a graph representing the oleic acid conversion rate according to thepenetration time of the mixture through the porous membrane having theimmobilized enzyme prepared according to an exemplary embodiment of thepresent invention at 25 and 37° C. It was confirmed that at 25° C., theoleic acid conversion rate was maintained at about 37% even for themixture penetration time of 300 hours or more, and at 37° C. which isknown to be an optimal activity temperature of the lipase enzyme, theoleic acid conversion rate was significantly increased to maintain atabout 63%. Hereby, the stability of the immobilized enzyme may beverified.

Further, the porous membrane of the present invention is reusable, andFIG. 10 is a graph representing an oleic acid conversion according tothe number of reuse of the porous membrane prepared according to anexemplary embodiment of the present invention. The conversion rate was63% when the membrane was used once 50% when used twice, and 54% whenused three times. Each time the membrane was used continuously byflowing reactants for 300 hours and then stored in a refrigerator beforenext use. As the number of use is increased, the conversion rate wasslightly lowered as compared with the conversion rate of first use, butstill high as compared with the case without the porous membrane.

Example 2: Preparation of Biodiesel

Biodiesel was prepared using the porous membrane prepared according toExample 1. (a) A mixed solution of 1 mol of soybean oil and 4 mol ofethanol, (b) a solution formed by directly adding lipase to the mixedsolution of 1 mol of soybean oil and 4 mol of ethanol to cause reaction,and (c) a solution obtained by passing the mixed solution of 1 mol ofsoybean oil and 4 mol of ethanol through the porous membrane havingimmobilized lipase, were measured by a gas chromatography, and theresult is shown in FIG. 7. As shown in FIG. 7, it was confirmed thatmaterials in the form of fatty acid ethyl ester which is usable asbiodiesel were produced in the cases of the solution passed throughporous membrane having immobilized lipase of the present invention ((b)in FIG. 8) and the solution to which lipase was added ((c) in FIG. 8),but not in the case of the absence of lipase ((a) in FIG. 8). This meansthat lipase acted as a transesterification reaction catalyst.

Further, the spectrum peak intensity of solution (b) penetrating theporous membrane of the present invention was higher than that ofsolution (c) to which lipase was added. This means that the catalyticactivity of the porous membrane having immobilized lipase of the presentinvention is higher, and such result was generated from the fact that ifthe reactants are mixed directly with lipase, lipase is not mixed wellwith the reactant, so that the reaction is generated only in a limitedboundary surface, whereas if lipase is immobilized within the porousmembrane through which the reactant is passed, the contact area of thereactant and lipase is increased, thereby more activating the catalystreaction.

The porous membrane having an immobilized enzyme of the presentinvention has a high immobilization rate of the enzyme, caused by havinga large surface area, and low resistance to mass transfer, caused by thefact that due to a three-dimensional pore structure through whichreactants are movable, and thus, the immobilized enzyme has excellentcatalytic activity.

The porous membrane having an immobilized enzyme of the presentinvention are highly stable against both organic or aqueous solventbecause the pore structure are built upon a three-dimensional covalentnetwork and thus the pore dimension and shape is not swollen orcollapsed by the chemicals.

What is claimed is:
 1. A porous membrane having an immobilized enzyme,wherein the porous membrane is three-dimensionally interconnected bypores, the pores have a size of 5 nm to 100 nm, a framework of theporous membrane is a three-dimensional network formed by polymerizationof a first monomer and a second monomer, wherein the first monomer istetrakis(4-aminophenyl) methane (TAPM), and the second monomer ishexamethylene diisocyanate (HDI), and the three-dimensional network isformed by polymerization occurring by reacting the first monomer withthe second monomer.
 2. The porous membrane of claim 1, wherein theporous membrane has a thickness of 10 μm to 100 μm.
 3. The porousmembrane of claim 1, wherein the porous membrane has a structure of aflat membrane or a hollow fiber membrane.
 4. The porous membrane ofclaim 1, wherein the enzyme is one or more selected from the groupconsisting of lipase, amylase, protease, trypsin, papain, brinase,peroxidase, horseradish peroxidase (HRP), carbonic anhydrase, aquaporin,chymotrypsin, subtilisin, soybean peroxidase, chloroperoxidase,manganese peroxidase, tyrosinase, laccase, cellulase, xylanase, lactase,sucrase, organophosphohydrolase, chlorinesterase, glucose oxidase,alcohol dehydrogenase, glucose dehydrogenase, hydrogenase, and glucoseisomerase.
 5. A multiple layered membrane having a structure wherein twoor more of the porous membranes of claim 1 are overlaid.